twenty odd years of stretch-sensitive channels · or osmotic pressure gradients applied across the...

INVITED REVIEW

Twenty odd years of stretch-sensitive channels

O. P. Hamill

Received: 14 June 2006 /Accepted: 27 June 2006 / Published online: 21 September 2006# Springer-Verlag 2006

Abstract After formation of the giga-seal, the membranepatch can be stimulated by hydrostatic or osmotic pressuregradients applied across the patch. This feature led to thediscovery of stretch-sensitive or mechanosensitive (MS)channels, which are now known to be ubiquitouslyexpressed in cells representative of all the living kingdoms.In addition to mechanosensation, MS channels have beenimplicated in many basic cell functions, including regula-tion of cell volume, shape, and motility. The successfulcloning, overexpression, and crystallization of bacterial MSchannel proteins combined with patch clamp and modelingstudies have provided atomic insight into the working ofthese nanomachines. In particular, studies of MS channelshave revealed new understanding of how the lipid bilayermodulates membrane protein function. Three major mem-brane protein families, transient receptor potential, 2 poredomain K+, and the epithelial Na+ channels, have beenshown to form MS channels in animal cells, and theirpolymodal activation embrace fields far beyond mechano-sensitivity. The discovery of new drugs highly selective forMS channels (“mechanopharmaceutics”) and the demon-stration of MS channel involvement in several major humandiseases (“mechanochannelopathies”) provide added moti-vation for devising new techniques and approaches forstudying MS channels.

Keywords Mechanosensitive channels .

Mechanotransduction . Transient receptor potential .

Patch clamp . Giga seal

Introduction

The formation of the giga-seal depends upon applyingsuction to draw the membrane into the pipette. Once theseal is formed, suction is not necessary, and it should bereleased [53]. However, the membrane patch spanning thepipette can now be mechanically stimulated by hydrostaticor osmotic pressure gradients applied across the patchwithout disrupting the seal. This feature allowed the firstrecordings of cell-swelling and stretch-activated channelcurrents [50, 52]. Over the last 20-odd years, interest inmechanosensitive (MS) channels has progressed from beinga possible patch clamp recording artifact to a central playerin our understanding of protein–bilayer interactions and apromising new therapeutic target against several majorhuman diseases. This article highlights some recent devel-opments and unresolved issues regarding MS channels,with a major focus on the MS Ca2+ permeant cationchannel (MscCa) recently identified in vertebrate cells [97].

What happens to the membrane patch in the pipette?

An important issue for MS channels is how the process ofaspiration and sealing of the membrane in the pipettealters the mechanics and possible stretch sensitivity ofchannels in the patch. Because of the small size andinaccessibility of the patch in the pipette, a variety oftechniques, including high-resolution video imaging [121,152–154, 184], high-voltage electron microscopy [142],atomic force microscopy [70], and fluorescence-imagedmicrodeformation [32, 33] have been used to study theaspirated patch and its underlying cell cytoskeleton(CSK). Here we focus on results obtained on the Xenopusoocyte [182–185]. The first issue is whether the pressure/suction applied to the patch after seal formation somehow

Pflugers Arch - Eur J Physiol (2006) 453:333–351DOI 10.1007/s00424-006-0131-0

O. P. Hamill (*)Neurosciences and Cell Biology, UTMB,Galveston, TX 77555, USAe-mail: [email protected]

induces changes in the seal resistance that appear as “MS-channel-like” events. This idea is not entirely far-fetchedbecause video imaging indicates that suction tends to peal themembrane off the walls of the pipette [121], and gated,cation-selective channels have been recorded with patchpipettes sealed onto noncellular hydrophobic surfaces [145].However, although strong suction can rupture the patch, ittypically does not disrupt the giga-seal, thereby allowing fortight seal whole-cell and/or outside-out patch recording [53].In addition, suction ramps applied to cell-attached frogoocyte patches reversibly activate either a saturating macro-scopic current (Fig. 1a) or a unitary amplitude current event(Fig. 1b) depending upon patch area [60]. These currentwaveforms are consistent with multiple and single MSchannel patches, respectively, but difficult to reconcile withMS changes in seal resistance. Even more compelling is thatpatches formed on pure liposomes fail to express MSchannel currents [97, 102, 122, 160]. This absence allowedfor the identification of MS channel proteins followingfunctional reconstitution of solubilized membrane proteinsfrom bacteria and archaea [81, 158, 159] and, most recently,from Xenopus oocytes [97].

Although MS channels are clearly not seal “leaks,” thesealing process does change patch geometry and theunderlying CSK, thereby altering patch mechanics. Forexample, Fig. 2a and b shows electron microscopy (EM)

images of the Xenopus oocyte surface, indicating itsextensive membrane folding and high density of microvilli(est. ∼7 microvilli per square micrometer) [181, 184, 185].This complex membrane geometry is also reflected inelectrical capacitance (Cm) measurements that indicate amembrane surface area that is tenfold greater than thatrequired for the cell’s volume [184]. In contrast, high-resolution video images of cell-attached oocyte patches(Fig. 2c, d) indicate an optically smooth membrane that ispulled flat and perpendicular to the walls of the pipette[184; see also 113, 152, 153, 161]. Furthermore, Cm

measurements indicate a patch area of approximately50 μm2, consistent with the patch geometry but inconsistentwith the approximately 500 μm2 expected if the approxi-mately 300 microvilli and membrane folds evident inFig. 2a and b were preserved during the sealing process[184]. Presumably, the suction used to obtain the giga-sealis sufficient to smooth out the surface folds and microvilliso that the cell membrane is now tightly stretched over anexpanded CSK (Fig. 3). This smoothing out of microvilli isnot an exclusive patch clamp phenomenon because asimilar phenomenon has been visualized in EM studies ofcells undergoing osmotic inflation [82] and spreadingbefore cell migration [37]. In these cases, the process ispresumably reversible, indicating the considerable plasticityof the microvilli and their supporting CSK.

There are at least two related mechanisms by whichchanges in patch geometry will increase stretch sensitivityof channels in the patch. First, in the absence of any excessmembrane, brief pressure pulses applied to the patch willrapidly flex the membrane and increase bilayer tension(Fig. 2c, d). The flexing of the membrane either outwardwith suction or inward with pressure results in the rapidactivation (<1 ms) of inward channel currents [107, 108,184]. In contrast, more sustained pressures would berequired to inflate the oocyte and smooth out the membranereserves to increase membrane tension (Tm) [15, 117, 184].Second, according to Laplace’s law, P=2 Tm/r, the pressure(P) required to activate channels in the patch with a radiusof curvature (r) of approximately 1 μm would be 1/20th ofthat required to activate the same channels located onmicrovilli that have a radii of curvature of approximately0.05 μm. For example, stimulus–peak current relationsshown in Fig. 4 indicate that half the channels in an oocytepatch are activated by a suction of approximately 10 mmHg(∼1.3 kN m−2), which translates to a tension of approxi-mately 0.6 mN m−1 (i.e., the near-symmetrical suction/pressure relations indicate a tension-gated channel andjustify Laplace’s law). To achieve the same tension inmicrovilli would require a suction of 200 mmHg. However,this would exceed the approximately 100-mmHg thatcauses patch rupture (i.e., a lytic tension of ∼6 mN m−1)under these conditions [119].

Fig. 1 Ramps of suction applied to two different-size patches formedon a Xenopus oocyte are consistent with a finite number of discreteMS channels but inconsistent with pressure-induced leaks in the seal.a A relatively large patch formed with an approximately 3-μm-diameter tip pipette shows a current that fully saturates at around40 mmHg. The arrow indicates the initial opening of 5 pA singleMscCa. b A smaller patch formed with an approximately 0.50-μm-diameter tip pipette reveals the opening of single MscCa, indicatingthat once open, the channel current was independent of suction. Theseresults indicate that MscCa is either open or closed, and the saturationof current in a multichannel patch represents the balance betweenopening and closing rates for a finite number of channels

334 Pflugers Arch - Eur J Physiol (2006) 453:333–351

The sealing process may also alter patch mechanics bychanging the CSK structure underlying the patch [55, 63,173]. When a gentle sealing protocol is used to achievethe giga-seal, the cortical CSK network that is pulled into thepipette may be dilated without disrupting its links withthe membrane [54, 185; see also 32, 33]. However, ifthe suction used to draw the membrane into the pipetteexceeds the strength of CSK–membrane linkages, then aCSK-free membrane (bleb) may be formed [55, 63, 112].Similarly, after the membrane has sealed in the pipette,additional suction may cause the blebbing at the membranecap as shown in Fig. 5 [185]. This membrane blebbing caneither increase or decrease the stretch sensitivity depending

upon the specific MS channel. For example, the TREK andTRAAK MscK channels show an increase in stretchsensitivity, presumably because the CSK normally acts as aconstraint and prevents tension being conveyed to the bilayer[69, 151]. On the other hand, MscCa typically shows a lossof both stretch sensitivity and fast dynamics presumablybecause they depend upon CSK interactions with thechannel/membrane (Fig. 5) [55, 156, 185].

In summary, giga-seal formation introduces significantchanges in patch mechanics that can alter the mechano-sensitivity of channels in the patch. The extrinsic changesin membrane geometry and CSK structure may havedifferent effects on specific channels depending upon

Fig. 2 Comparison of themembrane geometry of theoocyte surface and of the patchsealed in a pipette. a Transmis-sion EM of the oocyte surfaceshowing prominent microvillicontaining dark cytoplasmicmaterial. b Scanning EM of theoocyte surface, indicating thehigh density of microvilli.c High-resolution video imagesof a membrane patch before(0 ms), during (50 ms), and after(250 ms) a 100-ms suction step.d The same patch as in C excepta 100-ms pressure step wasapplied. Both suction and pres-sure steps activated a 50-pAinward current (modified fromZhang and Hamill [184] andZhang et al. [185] withpermission)

Pflugers Arch - Eur J Physiol (2006) 453:333–351 335

their intrinsic properties (i.e., protein structure and protein–CSK interactions).

Do animal cells generate/experience membrane tensionsthat activate MS channels?

Although it has been questioned whether animal cellsexperience the same in-plane membrane tensions thatactivate MS channels in the patch [30, 149, 172], there isevidence that they can experience even larger tensions thatresult in membrane rupture. In particular, cell woundingevents, as judged by the cell filling with the membraneimpermeant fluorescein-labeled dextran, have been ob-served in experimentally unperturbed rodent skin, gutepithelium, cardiac, and skeletal muscle, and found toincrease in frequency with mechanical loading [11, 109–111, 164]. The cell types that commonly experiencewounding in vivo include epidermal cells and fibroblasts(skin), epithelial cells and smooth muscle (GI tract andrespiratory system) endothelial cells, and smooth muscle(cardiovascular system) and peripheral neurons. The pro-portion of the cells wounded in the various systems canrange from 2 to as high as 25%. In eccentrically exercisedmuscle (e.g., downhill running), there can be a tenfold

increase in cell wounding compared with resting muscle,and in dystrophic mice that lack the CSK–structuralprotein dystrophin, exercise can produce massive wound-ing events that ultimately overload the muscle regene-ration mechanisms [180]. Normal migrating cells cangenerate traction forces that not only lengthen and stretchthe cell but also cause cell fragments to be ripped off anddeposited along the migration trail [104]. Furthermore, ifthe normal contractile mechanisms that allow a migratingcell to retract its rear are blocked, the front of the cell cantear away from the cell body and move off as a motile cellfragment [170]. The common occurrence of these trau-matic mechanical stresses under physiological and path-ological conditions has presumably provided strongselective pressure for the evolution of the membraneresealing mechanism(s) that is widely expressed ineukaryotic cells [11, 109–111, 164].

Despite the above evidence of membrane rupturetensions (i.e., >5 mN m−1), direct estimates of membranetension in “resting” isolated cells indicate much lowervalues (<0.1 mN m−1) [28–31, 116, 138]. The tensionmeasurement involves pulling a tether from the cell surfaceusing optical tweezers and measuring the force required tomaintain it at constant length. The basic assumption is thatmembrane tension is contiguous over the whole surface sothat pulling a tether from one region perturbs the tension inall regions of the cell membrane. From the static tetherforce (F0) measured in piconewtons, one can estimate themembrane tension Tm according to the equation:

F0 ¼ 2π 2BTmð Þ1 2=

where the membrane bending stiffness (B) is assumed to beconstant with a value of 2.7×10−19 N m−1. The practicallimitation of this technique is that the optical tweezers canonly sustain forces up to 100 pN, which would correspondto a tension of 0.5 mN m−1. For an animal cell with itscortical CSK, the measured tension is assumed to representa combination of in-plane tension and CSK adhesion, and isreferred to as the “apparent” tension. However, Tmmeasurements of membrane blebs that lack CSK indicatethe in-plane tension contributes only 25% of the Tm value[28]. Experiments on two different cell types (RBL 2H3cells and snail neurons) indicate that cell swelling increasessteady-state tensions from approximately 0.04 to 0.12mN m−1,which then returns to approximately 0.04mN m−1 with reshrinking [30, 31]. However, the samecells also experience tension surges that exceed the strengthof the trap (i.e., >0.5 mN m−1). Based on other experimentsmeasuring exocytosis/endocytosis as a function of mem-brane tension, it has been proposed that membrane surfacearea and tension are in a feedback loop in which hightensions favor membrane recruitment, and low tensionfavors membrane retrieval [31, 116, 148]. As a conse-

Fig. 3 Schematic illustrating the proposed smoothing out of micro-villi caused by the pipette aspiration and giga-seal formation


quence, it has been proposed that surface area regulation(SAR) maintains membrane tension around a relatively lowset point of approximately 0.1 mN m−1, which would bewell below the lytic tension (≥5 mN m−1) and the near-lytictensions (∼4 mN m−1) required to activate the bacterial MSchannels. However, the direct measurement of high tensionsurges exceeding the low set point and the occurrence ofcell wounding events indicate that SAR mechanisms can besaturated. Furthermore, as indicated in Fig. 6, lowertensions are required to activate MS channels in animalscells (T50% for MscK=2.4 mN m−1) and (MscCa ∼0.6mN m−1) compared with MscL (∼4.7 mN m−1) thatfunctions as a last-resort safety valve [94]. As a conse-

quence, MS channels in animal cells would seem moregeared to regulating processes with lower tension set pointssuch as regulatory volume decrease [20, 26, 146, 147, 169]cell locomotion [92] and, possibly, SAR via Ca2+-inducedexocytosis. However, it appears that integrins rather thanMscCa act as the mechanosensor for MS exocytosis/membrane trafficking at the frog neuromuscular junction[24] and the oocyte [96].

“Mechanopharmaceutics”

Progress in the MS channel field would be greatlyenhanced by the discovery of high-affinity agents that

Fig. 4 Inward current responsesto suction and pressure pulsesapplied to an oocyte patch.a Both suction and pressurepulses (2.5 s) result in rapidopening of MscCa that mostlyclose within 200 ms of thepulse. b Schematic showingflexion of the patch outward(suction) or inward (pressure).c Stimulus–response relationsfor suction and pressure steps.The sigmoid fits indicate thatsuction (P0.5=−10 mmHg) wasslightly more effective thanpressure (P0.5=14 mmHg) inactivating MscCa


selectivity target specific MS channels. These agents wouldbe highly useful for following MS channel proteins duringpurification procedures and identifying MS channel roles innovel functions. In addition, given that MS channels maybe polymodally activated (e.g., by tension, voltage, pH,temperature, Ca2+ store depletion, and/or lipid secondmessenger) [27, 125, 171], it would be advantageous todiscover agents that selectively acted on MS channel onlywhen they were mechano-gated. Although no ideal MSchannel reagent has yet been discovered, a number ofcompounds have been identified that act as MS channelblockers or activators [48, 61, 65]. One class, amiloride andits analogs, appear to act on a traditional “lock and key”protein receptor, whereas other agents, GsMTx-4 andpossibly maitotoxin, seem to act via nontraditional “recep-tors” at the lipid or lipid–protein interface where they may

change the local bilayer mechanics and thereby modify MSchannel gating. Below we briefly review their salientfeatures.

Amiloride has been the most rigorously studied in termsof its MS channel blocking mechanism and provides anexample where variations in mechanistic detail may enablediscrimination between different channel families in termsof their participation in specific MS functions. In particular,the amiloride block of MscCa/TRPC-1 in Xenopus oocytes[56, 89–91] and MscCa in vertebrate hair cells [141] hasbeen shown to involve basically the same unusual mech-anism in which two amiloride molecules bind cooperativelyto channel sites that only become accessible at hyper-polarized potentials after the channel has opened. Thismechanism, referred to as “conformational” block, impliesdifferent open-state conformations at hyperpolarized vs

Fig. 5 Changes in the membrane and patch currents as a consequenceof repetitive pressure pulses applied to the patch pipette. a–d videoimages of a cell-attached patch at different times after formation of thegiga-seal. Between each image, suction steps (20 mmHg, 500 ms)were applied. a The first image taken immediately after giga-sealformation shows the patch curved outwards and located closed to theend of the pipette (∼5 μm). Particles located in the cytoplasmexhibited no motion presumably because they were still constrainedby the intact CSK. b–d repetitive suction pulse caused the patch to

move up the pipette away from the cell, and a clear space developedbetween the membrane and the CSK remaining close to the cell.Particles that moved into this space displayed Brownian motion,indicating the loss of constraining CSK structures [153]. e Applicationof suction pulses at a caused a rapid opening of MscCa that closedalmost completely. f Application of a suction pulse at d causedactivation of a smaller more sustained current (from Zhang et al. [185]with permission)


depolarized potentials, and is distinctly different from theamiloride block of the high-affinity amiloride-sensitiveepithelial Na+ channel (ENaC) where the voltage depen-dency arises because the positively charged amiloride binds

to a pore site that senses a fraction of the electric field[130]. A further difference is seen in the order of potenciesof amiloride analogs in blocking the two channel classes(Table 1) [80]. Amiloride blocks the MEC-4 DEG/ENaCcurrents in touch receptor neurons in Caenorhabditiselegans [120] and also the mammalian arterial myogenicresponse, which has been used to implicate DEG/ENaC asthe vascular mechanosensor [34]. However, amiloride alsoblocks TRPC-6, also implicated as the arterial smoothmuscle mechanosensor [74, 175]. It will be interesting todetermine if mechanistic differences in amiloride block(i.e., conformational vs pore block) can be used to resolvethe MS Channel_s identity.

Gadolinium blocks a variety of MS channels (e.g.,MscCa, MscK, MscL, and MscS), several TRP channels(e.g., TRPC-1, TRPC-4, TRPC-5, and TRPV1), variousvoltage-gated (Ca2+, Na+, and K+) and receptor-gatedchannels [e.g., N-methyl D-aspartate (NMDA), AChR,etc.] [61, 124, 136, 144, 165, 176, 178]. Because of itstrivalency, Gd3+ will bind with high affinity to negativegroups on proteins, lipids, and polysaccharides, as well asany inorganic anions present in solution [17, 61]. Its ionicradius (0.938 Å), which is similar to Na+ (0.97 Å) and Ca2+

(0.99 Å), may also allow it to enter and bind to negativelychange groups (Glu and/or Asp) within cation channels.Evidence that Gd3+ interacts directly with channel proteinscomes from studies of specific TRP members (TRPV-1,TRPC-4, and TRPC-5) where Gd3+ has been shown to havedual effects, activating the channels at low micromolarconcentrations (<100 μM) but blocking at higher concen-trations (>300 μM). The activation of TRPV-1 dependsupon binding to specific external glutamate residues thatconfer acid sensitivity on the channel, and neutralization ofthese residues blocks the activation and modifies inhibition[165]. Similar concentration-dependent potentiating andblocking effects also occur with TRPC-4 and TRPC-5[77]. In contrast, Gd3+ only blocks TRPC-1 and TRPC-3channels at relatively low micromolar concentrations [97,166].

0 20 40 60 80 100

mmHg

0

0.5

1.0

1.0

0.5

0

0

0.5

1.0

MscL

79 mmHg

MscK

40 mmHg

MscCa

10 mmHg

Fig. 6 Comparison of the normalized pressure–current relations forthree different MS channels. MscCa curve fitted to curve Fig. 2c (top).MscK curve fitted to data from Ref. [69] (middle). MscL curve fittedto data from Ref. [54] (bottom)

Table 1 Amiloride analog potency (IC50 amiloride/IC50 analog) of MS channels and the epithelial Na+ channel

Amiloride(IC50, μM)

DMA Phenamil PBDCB Benzamil HMA I-NMBA Reference no.

MscCa mouse hair cell 1 (53) 1.3 4.4 5 9.6 12.3 29 [141]MscCa Xenopus oocyte 1 (500) 1.4 – – 5.3 14.7 (BrHMA) [90]Epithelial Na+ channel(high affinity)

1 (0.34) 0.04 17 – 9 0.04 7 [80]

Epithelial Na+ channel(low affinity)

1 (10) 2.2 2.9 – 3.8 – – [80]

DMA Dimethylamiloride; PBDCB 5-(N-propyl-N-butyl)-dichlorobenzamile; HMA hexamethyleneamiloride; I-NMBA 6-iodide-2-methoxy-5-nitrobenzamile; BrHMA bromohexamethylene amiloride


An early view on how Gd3+ might block MS channelswas via effects on the bilayer. Gd3+ has been shown tointeract with black lipid membranes containing the nega-tively charged phosphatidylserine (PS) but not with theneutral phosphatidylcholine to increase the boundarypotential and membrane tension [38]. However, whetherthese effects underlie MS channel block remains unclearbecause PS is normally restricted to the internal-facingmonolayer, and Gd3+ acts externally. Gd3+ has also beenshown to promote shape changes in giant unilamellarvesicles lacking PS [162]. In this case, it was proposedthat Gd3+ bound to the hydrophilic lipid head (i.e., negativecharge of the phosphate groups) of the external monolayer,and in doing so, decreased its surface area relative to theinternal monolayer, thereby causing a change in mem-brane curvature. However, whether this effect wouldblock MS channels remains unclear because amphipathsthat also change membrane curvature usually result in MSchannel activation [101, 135]. A recent study has reportedthat Gd3+ can block MS channels without alteringpressure-induced changes in Cm, which would beexpected if Gd3+ acted by altering membrane mechanics[156]. However, as pointed out by the authors, measure-ment of this parameter may be complicated because Gd3+

has multiple effects, including increasing the giga-seal[35].

GsMTx-4 is a 34-amino acid (4 kDa) peptide isolatedfrom tarantula venom [48, 49, 155, 157]. It is amphipathicwith a hydrophobic membrane face opposite a positivelycharged face, and it is a member of the inhibitory cysteineknot (ICK) toxin superfamily. GsMTx-4 is the most specificMS blocker identified to date. Because of its structure, itwould be expected to be attracted to negative regions ofproteins/lipids, and it will tend to partition into hydrophobicpockets either within the protein or at the protein/lipidinterface. Unlike the nonspecific channel blocker Gd3+,GsMTx-4 has so far not been reported to affect voltage- orreceptor-gated channel. GsMTx-4 blocks MscCa at between0.2 and 3 μM in chick heart, rat astrocyte, and humanbladder and kidney cells [48, 49], and the crude tarantulavenom also blocks MscCa in growing pollen protoplasts[36]. Most recently, GsMTx-4 has been shown to stimulateneurite outgrowth by blocking Ca2+ elevation in Xenopusspinal neurons [76]. However, GsMTx-4 does not blockMscCa involved in auditory transduction [48], MscKformed by TREK (E. Honore, unpublished observations,cited in Ref. [48]) the bacterial MscL [99], and perhaps,most surprisingly, MscCa in Xenopus oocytes (R. Marotoand O.P. Hamill, unpublished observations). The last resultis puzzling given that the oocyte MscCa is often treated asthe prototypical MscCa, and TRPC-1 has been implicatedas forming MscCa in the Xenopus oocyte and mammaliancells [97]. One possible explanation is that there are

structural differences between MscCa proteins in differentcell types. However, based on the observation that GsMTx-4synthesized from D instead of L amino acids shows the samepotency in blocking specific MS channels, it has beenproposed that GsMTx-4 is more likely to act by binding toboundary lipids surrounding the channel, and, at least,consistent with this is that GsMTx-4 and its enantomer alsoalters the gating of gramicidin A, which is particularlysensitive to bilayer mechanics [157]. It may therefore be thatdifferences in lipids between poikilotherms vs homeothermsis a factor that underlies the different GsMTx-4 sensitivities.In this case, it will be particularly interesting to determineGsMTx-4 action on MscCa/TRPCs reconstituted into de-fined lipid environments.

Maitotoxin (MTX) is a highly potent marine poison (LD50 for mice 50 ng/kg) from the dinoflagellate (Gambier-discus toxicus) that is responsible for Giguartera, a formof seafood poisoning [40]. It is water-soluble polyetherwith 2 sulfate esters, 28 hydroxyls, and 32 ether rings, andwith a molecular weight of 3.4 kDa, it is the largestamong the known nonbiopolymers. The hydroxyl andionized sulfate groups makes MTX a highly polarsubstance, but the presence of large hydrophobic portionsmake it amphipathic so that it most likely inserts itselfdeep into the bilayer. MTX elicits Ca2+ influx in a varietyof cell types, and the Ca2+ influx may lead to secondaryeffects, including phosphinositide breakdown and arachi-donic release. Of special interest here in that cellsexpressing TRPC-1 show a substantial increase in MTX-initiated Ca2+ influx that is blocked by Gd3+ (KD50=3 μM)and also by amiloride and benzamil but not by flufenamicacid or niflumic acid [10, 14, 174]. MTX activates 40 pSchannels when applied to outside-out patches butnot inside-out patches indicate that MTX acts on theextracellular face and does not require second messengers[40]. Both the conductance and pharmacologicalproperties have led to the idea that MTX activates theMscCa channels in oocytes, which is consistent withits effect of activating similar channel currents inTRPC-1 expressing cells. On the other hand, MTX alsoincreases Ca2+ influx in red blood cell (RBC) ghostswhich may involve another mechanism [85]. Although ithas been suggested that MTX mainly acts to increasecurrent by stimulating insertion of channels in the oocytemembrane, the evidence is based on large rapid Cm

changes that follow moment-to-moment changes inconductance induced by MTX and which are blocked bythe same ions and agents that also block the conductancechanges [174]. These properties indicate the Cm changesmay have been contaminated by changes in membraneconductance [25]. In this case, alternative methods formeasuring membrane trafficking (e.g., FMI-43 fluores-cence) should be used to test whether MTX-induced


membrane conductance occurs by channel insertion and/or channel activation [40].

Mechanosensitive channel protein identification

The membrane proteins forming specific MS channelshave only been recently identified, and there were severalreasons for this delay, including the general low abun-dance of MS channels in animal cells, the absence ofhigh-affinity MS channel agents, the inability to employconventional expression cloning strategies because ofwidespread endogenous MS channel expression, and theabsence of identified mutant phenotypes involvingstretch-activated channels. To overcome these handicaps,a novel strategy was developed by Sukharev et al. [159,160] that involved detergent-solubilizing and fractionatingmembrane proteins, reconstituting the protein fractions inliposomes, then assaying the fractions for stretch sensitiv-ity using patch clamp recording. This technique has beenused to identify a variety of MS channel proteins inbacteria and archaea [81, 103, 158, 159] and, mostrecently, the TRPC protein family in forming MscCa inXenopus oocytes [97]. Furthermore, by demonstrating thata purified protein reconstituted in pure liposomes can retainstretch sensitivity, the technique also provided unequivocalevidence for the idea that bilayer tension alone gated MSchannels [101]. Although ENaC has also been reconsti-tuted in lipid bilayer and reported to show stretchsensitivity, it is not clear whether the proposed mechanismof stretch-induced release of Ca2+ channel block operatesin situ [75]. At this time, the best evidence for ENaC familymembers forming MS channels comes from genetic studiesof C. elegans touch-insensitive mutants [see 8, 39, 47, 62for reviews].

The proteins forming the other major class of MSchannels in animals cells, MscK [79, 118, 150, 168], wereidentified serendipitously, in that after the first membersof the 2 pore domain K+ (K2P) channel family hadalready been cloned and shown to form K+ channels [41,42, 93], they were subsequently found to be stretch-activated [6, 126, 129]. The recent demonstration thatTREK and TRAAK retain stretch sensitivity in CSK-freemembrane blebs indicates that they are also bilayer-gatedchannels [69]. In addition to the MS channel proteinsthat may function as mechanosensors in situ, there isalso an increasing number of voltage-gated and receptor-gated channels as well as peptides that form simplemodel channels (alamethicin and gramicidin) that displaymechanosensitivity [19, 102, 115, 122, 163]. Althoughthese channels may operate on the same general princi-ples that confer mechanosensitivity on membrane pro-teins, their role, if any, as mechanotransducers remains tobe demonstrated.

Mechanosensitive channel dynamics: adaptation/desensitization/inactivation

Gating dynamics (adaptation/inactivation/desensitization)has been shown to play a critical role in the signaling byvoltage- and receptor-gated channels and the hair cellmechanotransduction channel [67, 72] and defects in gatingdynamics underlie a number of channelopathies [4]. In theinitial studies of single MS channel currents, the channelsseemed to obey stationary kinetics and were analyzedaccordingly [46, 50, 51, 90, 114, 143, 177, 179]. However,with the ability to apply fast pressure steps to the patch [9,59, 105–108], it became evident that MS channels alsodisplayed dynamics in which the channels either closedreversibly (adaptation or inactivation/desensitization) orfaded irreversibly (run down) with constant stimuli [55,69, 105, 106, 156].

In principle, the reversible closure of MS channelsduring maintained stimulation can arise through relaxationin either the mechanical force being applied to the channelor the sensitivity to that mechanical force [54, 57] Becausemechanical gating arises from the channel protein beingsensitive to some mechanical-induced deformation [i.e.,either in the bilayer or in CSK/extracellular matrix (ECM)elements], then closure can arise because of a relaxation inthe force causing the deformation or a relaxation in thesensitivity to that deformation. For example, in the simplestcase of a two-state channel in which the rate constants forchannel opening (β) and closing (α) are displacement-sensitive (i.e., for a tethered MS channel) or tension-sensitive (i.e., for bilayer-gated MS channel) the probabilityof the channel being open (Po) will be given by:

Po ¼ 1 1þ Kð Þ=

where

K ¼ β a=

Or, in terms of displacement,

K ¼ K0es x0�xð Þ

where K0 is the equilibrium constant when the displacementx is equal to the set point x0 and determines the number ofchannels open at zero relative displacement, and s is thesensitivity to the relative displacement change (x0−x). For abilayer-gated channel, we can substitute displacement witharea change. An exponential time relaxation in either s or x0can produce the same adapting MS channel currents [57].Figure 7 illustrates simulations of the stimulus-responserelations made, assuming that after a step stimulus, there isan exponential change in either the set point x0 (Fig. 7a) orthe sensitivity factor s (Fig. 7b).

Although both mechanisms predict the same kinetics ofchannel closure, the consequences on the Po−X curves are


clearly different. In the first case of adaptation, the curvesshift along the x-axis with no changes in slope (i.e.,sensitivity) around a common set point. In the second case,

the sensitivity decreases as the curves pivot around acommon point. From a functional point of view, the firstcase is true adaptation because sensitivity is maintained

Fig. 7 Simulation of two mechanisms that results in closure of MSchannels in the presence of sustained stimulation. A two-state channelis assumed, and a step displacement from 0 to x (top, trace 1) is usedto activate the MS channels (a and b). Trace 2 represents the changesin tension (a) or sensitivity tension (b). In trace 3, the channel currentsare represented by the change in open-channel probability (Po), withthe numbered points (1–5) representing equally spaced times wherePo–X curves were generated to follow changes in the MS channelsensitivity. a The decay of the current is due to a change in the tension

(measured as x−x0) caused by a shift in the set point. In this case, thereis a shift along the x-axis with no change in slope. Consequently, theδPo response due to δx does not decrease during what can beconsidered true adaptation. b The decay of the current is due to achange in sensitivity in which the slopes of the Po–X curves decreaseas they pivot around a common point. As a consequence, theincremental change in the response (δPo) for a fixed δx decreasesduring the current decay, which is akin to receptor desensitization.Modified from Ref. [57]


[55], whereas the second case is more akin to receptordesensitization or voltage-gated channel inactivation, wherethe stimulus must be removed for sensitivity to recover[69]. Below, we consider specific MS channels in terms ofthese general principles.

MscCa in Xenopus oocytes This channel displays differentgating dynamics depending upon patch “history.” In thecase when the giga-seal is formed using a “gentle” suctionprotocol (e.g., 10 mmHg for 1–10 s), the application of asuction/pressure step produces rapid opening (<1 ms) ofchannels followed by a slower closure (∼100 ms), althoughthe stimulus is maintained constant. The resultant decay ofMscCa current can be fitted by a single exponential with atime constant of around 100 ms at −100 mV that shows amonotonic e-fold increase for every approximately 100-mVdepolarization. The voltage dependence of the channelclosure is most evident when the voltage is switched fromhyperpolarized to depolarized potential (or vice versa)during the pressure step (Fig. 8) [55, 107, 108]. Thedirection of this voltage dependence is similar to thevoltage dependence of adaptation displayed by the hair cellmechanotransducer channel [5] and MscS (see below). Thestimulus induced closure of the oocyte channel wasoriginally referred to as adaptation because increasing thestimulus could reopen channels. In the oocyte, suctions/pressures of approximately 20 mmHg produce saturatingresponses (see Fig. 4), so it was assumed that any channelopening caused by an increase in suction/pressure of at least20 mmHg would involve reopening channels that had justclosed. However, a practical limitation in using theseprotocols on oocyte patches is that application of evenlarger pulses (e.g., ≥40 mmHg) that would undoubtedlyactivate all channels will also cause irreversible loss of thechannel activity and gating dynamics as described below[55, 105, 106].

In the second case, if a more forceful suction protocol isrequired to achieve the seal, then more often than not, thetransient current response is absent, and instead, thechannels remain open for the full duration of the suction.Similarly, if after a gentle seal is formed the patch ismechanically “over-stimulated,” then adaptation of MscCaactivity disappears either progressively with each moderate-sized pulse (Fig. 9a) or suddenly within a single largesuction (Fig. 9b). This transition from the transient mode(TM) to the sustained mode (SM) of gating is irreversibleand occurs without a change in single-channel conductance[64].

The fragility of MscCa dynamics and the transition fromTM to SM gating has been proposed to arise throughmechanical decoupling of CSK interactions with either thechannel or the membrane, which are thought to beimportant for TM of gating. It has been suggested that

viscous elements (dashpots) in the CSK can become frozenor decoupled without disconnecting the gating springs.However, adaptation is preserved in both inside-out andoutside-out patches, and in patches treated with agents thatdisrupt microtubules (colchicine) or microfilaments (cyto-chalasin D), similar to what has been reported for TRAAKdesensitization [69]. In contrast, transient gating kinetics ofMscCa are not retained in either “blebbed” membrane thatit lacks an underlying CSK [185] or in pure liposomepatches expressing MscCa activity following reconstitutionof detergent-solubilized oocyte membrane proteins [97].Furthermore, overexpression of TRPC-1 that forms theoocyte MscCa does not result in channel activity thatdisplays TM gating. Whether the absence of adaptationreflects prior mechanodisruption or the absence of CSKremains unclear, as does the mechanism that causesirreversible run down. One possibility is that there are

Fig. 8 Voltage dependence of pressure-induced currents recordedfrom cell-attached oocyte patches. In each panel, the top tracerepresents the pressure (suction); the middle trace, the voltage; andthe bottom trace, the current. a The application of the suction pulse tothe patch held at −100 mV caused rapid opening of channels that hadnearly all closed before the voltage was switched to 100 mV whilemaintaining the suction pulse. b In this case, the suction was appliedto the patch held 100 mV and produced a steady-state current.Switching the voltage to −100 mV activated a transient increase incurrent that decayed incompletely in the presence of maintainedsuction


irreversible changes in the membrane–glass adhesion thatalters the ability to generate tension changes in the bilayer.For example, if the membrane does not reseal aftermechanical decoupling of lipid–glass interface [121], thenincreasing pressure may draw further membrane into thepipette without increasing bilayer tension.

MscCa in rat astocytes This channel shows certain dynamicproperties similar to those of the oocyte MscCa, including itsvoltage dependence and mechanical fragility. However, inthe astrocyte, the decrease in current occurs because ofincreased occupancy of lower conductance states and areduced open-channel probability [13, 156]. Furthermore,the closed channels cannot be reactivated by increasing thestimulus strength (i.e., are refractory), indicating inactiva-tion rather than adaptation. The process was modeled as aball-and-chain-type inactivation, in which the inactivatingball was a CSK element rather than part of the channelprotein. By assuming that the binding rates of theinactivating ball were affected by the position of anintramembrane voltage-sensing subunit, one can accountfor the voltage dependence of inactivation. Apparentlyconsistent with the model, it was demonstrated that a

combination of agents targeting actin (cytochalasin), micro-tubules (colchicine), and intermediate filaments (acrylam-ide) was required to abolish the inactivation, but this lossmight again reflect general mechanical patch damage ratherthan implicating specific CSK elements. In the same study,fast Cm measurements were used to monitor the changein membrane area/thickness during pressure steps anddemonstrated a similar voltage-independent monotonicincrease in patch capacitance at −90 and + 50 mV, whichcontrasted with the voltage-dependent inactivation. Thisobservation was interpreted as indicating inactivation was dueto intrinsic properties of the channel rather than relaxation ofbilayer tension [156].

MscS and MscL in Escherichia coli The two predominantMS channels in Escherichia coli, MscS (0.5 pS) and MscL(1–3 nS) [100, 160], also exhibit transient gating dynamics[2, 66, 86, 87]. MscS currents measured in E. coliprotoplasts in response to pressure steps undergo apressure-induced exponential decay that appeared to bevoltage-independent with a time constant of 2–3 s whenmeasured over a narrow voltage range of ±30 mV [86].However, when measured over a wider voltage range of

Fig. 9 Irreversible loss of tran-sient mode gating of MscCa.a Three consecutive suctionpulses of 30 mmHg were ap-plied to a patch 30 s apart,causing a progressive loss of thetransient gating and a decreasein the peak current. b A singlelarge suction pulse (100 mmHg,10 s) was applied to a patch andcaused an initial peak currentthat was converted into a sus-tained current


±100 mV there was a decrease in the rate of inactivationwith depolarization similar to MscCa [2]. Application of thesecond of a double-step protocol activates fewer channels,and a finite time without stimulation is required forrecovery of the full response [86]. Furthermore, suctionramps produced smaller peak responses than suction steps[2]. These features are more consistent with inactivationthan adaptation. Unlike MscCa in vertebrate cells, MscSdynamics are not mechanically fragile, although pronasetreatment of the intracellular membrane face abolishes thetransient kinetics and, ultimately, mechanosensitivity. Thislast observation led Koprowski and Kubalski to proposethat both activation and inactivation may depend uponinteraction between a cytoplasmic (pronase-sensitive) re-gion of the channel with the lipid bilayer [86, 87]. Note theproteolytic inhibition of MscS activity is opposite to thepotentiation of MscL activity [1]. Given that a bilayer ratherthan a tethered mechanism gates MscS, it was proposed thatinactivation might be associated with insertion of thecytoplasmic domain of MscS in the bilayer (i.e., a “hybrid”or intrinsic tethered model; see below).

MscL reconstituted in liposomes also shows a transientdecay in the current with a time constant of seconds [66].Although the distinction between adaptation and inactivationstill needs to be made, the observation is significant becausethe clear absence of any CSK excludes its involvement inthese transient kinetics. One possible explanation is time-dependent sliding/relaxation of the two monolayers thatresults in relaxation of the gating tension [144].

TREK and TRAAK MS channels In a recent study, pressuresteps have been used to analyze the dynamics of MscKformed by cloned TREK-1 and TRAAK herologouslyexpressed in COS cells and Xenopus oocytes [69]. Bothchannels show rapid closure (τ∼20–50 ms), with constantstimulation similar to the MscCa. However, unlike MscCa,MscK gating dynamics are not voltage-sensitive, and eithermechanical or chemical disruption (i.., using latrunculin) ofthe CSK causes “run up” rather that “run down” of thechannels without removing the transient gating kinetics.Because it was clearly demonstrated that channels could notbe reactivated without a finite time for recovery, thephenomenon was referred to as desensitization. The lack

Fig. 10 Three different modelsof mechanosensitive channelgating a Bilayer. Mechanicalforces are conveyed to thechannel purely via the bilayer.Tension sensitivity occurs be-cause of a difference proteinarea (or hydrophobic thicknessand/or lateral shape) betweenthe open and closed channelconformations. b Extrinsic teth-er. Tensions are exerted directlyon the channel protein via ex-tracellular or cytoskeletal elasticelements/gating springs. Whentension is exerted on the gatingspring, the open state is ener-getically more favorable. Intrin-sic tether (hybrid). In this model,the gating spring is one of thecytoplasmic domains that bindsto the phospholipids and, in thisway, becomes sensitive tomembrane stretch


of effects of either mechanical or chemical CSK disruptionindicates that desensitization is an intrinsic property of thechannel [69].

Molecular models of stretch sensitivity

There are three broad classes of mechanisms that mayimpart stretch sensitivity on a membrane ion channel.They will be referred to as “bilayer,” “tethered,” and“hybrid,” and are shown schematically in Fig. 10. Themodels need not be mutually exclusive, and a singlechannel may derive its mechanosensitivity from all threemechanisms. Each mechanism can be discussed in termsof a simple two-state channel that fluctuates between aclosed and open state. The bilayer mechanism applies to avariety of MS channels, as evidenced by retention ofmechanosensitivity following liposome reconstitutionand/or activation by amphipaths or lysophospholipids.The basic idea is that stretching the bilayer will tend todecrease its lipid packing density and thickness, so that ifthe channel protein undergoes a change in membrane-occupied area (Fig. 9a) and/or hydrophobic mismatch,there will be a shift in the distribution between closed andopen channel conformations [54, 88, 95]. By inserting inthe membrane, lysophospholipids and amphipathic mole-cules may cause local changes in tension and curvature atthe lipid–protein interface and thereby shift the channeldistribution [12, 95, 98, 99, 131–133].

In the tethered mechanism, either an extracellular orcytoskeletal protein is directly connected to the channeland acts as a gating spring [50, 58, 62, 71, 72]. When thegating spring is stretched, it favors the open state of thechannel because it allows relaxation of the spring. In

Fig. 9b, the gate is represented as a trapdoor that opensout, but it can well represent subunits that are eitherpulled apart (increased in area) or lengthened (change ifhydrophobic mismatch). Evidence pro and con for thetethered mechanism has been discussed previously [54,88].

The hybrid of the above two mechanisms depends uponstretching of the bilayer, but in this case, there arecytoplasmic domains of the channel protein that bind tophospholipids, and in this way act as intrinsic tethers orgating springs that are stretched along with the bilayer(Fig. 9c). Evidence for the hybrid model comes from theidentification in the specific K2P channels of a phospho-lipid-sensing domain on the proximal carboxyl terminusthat involves a cluster of positively charged residues thatalso includes the proton sensor E306 [23, 68]. Protonationof E306 drastically tightens channel–phospholipid interac-tion and leads to TEK-1 opening at atmospheric pressure.The carboxy terminal domain of TREK-1 interacts withplasma membrane, probably via electrostatic interactionbetween a cluster of positive charges (a PIP2-interactingdomain) and anionic phospholipids.

Mechanosensitive channels in human diseases

An exiting development in the field has been the growingnumber of diseases associated with abnormalities ofmechanotransduction. Donald Ingber [73], in a recentreview, listed 45 diseases that may arise due to changes incell mechanics, alterations in tissue structure, or deregula-tion of mechanosignaling pathways. Of these diseases,several have been directly associated with changes inexpression and/or gating of MS channels, including cardiacarrhythmias [84], polycystic kidney disease [18], hyperten-

Fig. 11 Cell-attached patch re-cording on an mdx mouse myo-tube showing high constitutivechannel activity that was re-duced with suction but unaf-fected by positive pressure. This“apparent” stretch-inactivatedchannel activity was rare andseen in only 1 of approximately100 patches. The majority ofother patches showed no spon-taneous channel activity, andsuction activated either a tran-sient opening of channels orchannels that remained open for10 s after the pulse (e.g., see[107])


sion [83], glioma [123], glaucoma [78] atherosclerosis [22,134], Duchenne muscular dystrophy [44, 45], and tumor-igenesis [128]. Furthermore, increased MscK activity hasbeen shown to prevent brain ischemia [16] and promotegeneral anesthesia [127], whereas MscCa/TRPC activitymay regulate wound healing [137] and promote neuronalregeneration [76]. Of particular note is Duchenne musculardystrophy (DMD), a devastating X-linked genetic diseasethat affects approximately 1 in 3,500 male births and ischaracterized by progressive muscle wasting and weakness(reviewed in [180]). DMD is caused by the absence of thegene product of dystrophin, a cytoskeletal protein that bindsto actin and provides structural support for the membraneparticularly during muscle stretching. In mdx muscle fibers(i.e., from the mouse model of DMD), there is increasedvulnerability to stretch-induced membrane wounding, andseveral studies indicate elevated [Ca2+]i levels in mdxmyotubes that have been associated with increased Ca2+

permeant leak channel activity [43] and/or abnormal MscCaactivity [44, 45]. Anti-MscCa agents, including Gd3+,streptomycin, amiloride, and GsMTx-4, have been reportedto block Ca2+ elevation and/or reduce muscle fiberdegeneration [3, 56]. Based on the observation that theleak channel activity was increased by internal calciumstore depletion, Vandebrouck et al. [167] proposed that astore-operated Ca2+ channel (SOCC) belonging to theTRPC family may be involved. To test this idea, theytransfected muscles with antisense oligonucleotide designedagainst the most conserved region sequences of the TRPCsand showed it caused significant knockdown of TRPC-1and - 4 but not TRPC-6 (all three were detected in wild-type and mdx muscle), and reduced both control andthapsigagin-induced Ca2+ leak channels without affectingvoltage-gated Na+ channels. The mechanosensitivity of thechannels was not tested in this study. However, MscCa canshow significant spontaneous opening in the absence ofmembrane stretch [140]. Furthermore, although Franco andLansman [44] initially reported a stretch-inactivated Ca2+

channel in mdx mice, they subsequently concluded that thechannel activity may arise from a novel gating mode ofMscCa induced by membrane stress [45]. Most recently, ithas been suggested that stretch inactivation in patches ofmdx muscle and other cells may be a patch recordingartifact induced when suction applied to the patch reduces atonic tension generated by CSK forces that bend the patchtoward the cell [69]. At least consistent with this notion isthat suction (but not positive pressure) causes inactivationof MscCa in mdx patches (Fig. 11).

Conclusion and future prospects

The giga-seal patch clamp technique has been a majorcontributor to increased understanding of MS channels over

the last 20 odd years. However, there is still somewhat adisconnect between the phenomena seen in the patch andhow they translate in MS currents in the whole cell.Furthermore, given the growing evidence that MS channelsare promiscuous in terms of their modes of activation, itbecomes even more important to identify the exactphysiological stimulus that activates the channel in specificsituations. The development of new techniques that canmonitor/generate membrane tension changes in normallyoperating cells while recording MS channel on the cell canaddress many of the unresolved issues. Similarly, thediscovery of high affinity and selective agents that cantarget mechanically gated channels will represent a majorbreakthrough for the field. The determination of the crystalstructure of bacterial MS channels [7, 21, 139] has provideda rich environment for model building and testing, and asimilar trajectory is predicted for the recently identified MSchannel proteins in animal cells. A key question that thesestudies should answer is whether a unified set of principlescan account for the stretch sensitivity of channels in bothprokaryotes and eukaryotes [54, 88].

Acknowledgments Research in the author’s laboratory is funded bythe National Institutes of Health (NIH) and the Department of Defense(DOD).

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twenty odd years of stretch-sensitive channels · or osmotic pressure gradients applied across the...

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